Many aerobic microorganisms have a respiratory chain responsible for oxygen metabolism and can acquire energy in the presence of oxygen, and can be grown well in the presence of oxygen. Such aerobic microorganisms also have a mechanism of detoxifying superoxide anion or hydrogen peroxide, which may be generated from a portion of oxygen through metabolism thereof, with the aid of, for example, superoxide dismutase, catalase, or peroxidase, which enzyme eliminates the toxicity of such reactive oxygen species.
Lactic acid bacteria, which are known to be useful among anaerobic microorganisms, are facultative anaerobes, and are considered to have neither a respiratory chain nor catalase. However, many lactic acid bacteria can be grown even in the presence of oxygen and exhibit oxygen resistance. Hitherto, some studies have been conducted on the oxygen resistance mechanism of lactic acid bacteria and have shown that lactic acid bacteria have, for example, NADH oxidase or pyruvate oxidase as an oxygen-metabolizing enzyme. As has been reported, NADH oxidase is classified into two types, that is, water-forming type and hydrogen peroxide-forming type, and water-forming type NADH oxidase detoxifies oxygen by four-electron reduction to form water, whereas hydrogen peroxide-forming type NADH oxidase generates hydrogen peroxide by two-electron reduction. As has also been reported, in a lactic acid bacterium such as Streptococcus mutans, alkyl hydroperoxide reductase converts hydrogen peroxide into water, and these enzymes function as a two-component peroxidase.
In addition, some lactic acid bacteria have been reported to have, for example, superoxide dismutase, catalase, or NADH peroxidase, which eliminates superoxide anion or hydrogen peroxide generated from oxygen.
Studies have suggested that Lactobacillus plantarum WCFS1 exhibits enhanced resistance to oxidative stress such as hydrogen peroxide by enhancing expression of the thioredoxin reductase gene, and thus thioredoxin reductase plays an important role in resistance to oxidative stress (Non-Patent Document 1).
In Bacteroides fragilis, which is an anaerobic Gram-negative bacterium, only a thioredoxin-thioredoxin reductase system is considered to be responsible for redox reaction of thiol/disulfide. It has been reported that when thioredoxin reductase is deleted, the bacterium cannot be grown even under anaerobic conditions without addition of a reducing agent such as cysteine and dithiothreitol (Non-Patent Document 2).
In addition, as has been reported, Escherichia coli includes therein a glutathione-glutathione reductase system or a thioredoxin-thioredoxinreductase system, which is essential for maintaining the intracellular environment in a reduced state, and gene-disrupted strains involved in such a system are sensitive to oxidative stress including hydrogen peroxide.
As has been reported, growth of Lactococcus lactis is inhibited under aerobic conditions through disruption of thioredoxin reductase (Non-Patent Document 3). However, relation between growth inhibition of the bacterium and oxygen resistance thereof has not been elucidated, since growth of the bacterium under aerobic conditions is restored by addition of dithiothreitol, and the amount of cells of the bacterium after 24-hour culturing is nearly equal to that of wild type cells of the bacterium.
As has also been reported, a mutant strain of Streptococcus mutans obtained through knockout of both NADH oxidase and alkyl hydroperoxide reductase (ahpC) exhibits oxygen resistance, and thus the gene for another iron-binding protein is responsible for oxygen resistance (Patent Document 1). However, such a gene is not necessarily present in all microorganisms, and the mechanism of oxygen resistance has not yet been fully elucidated. As described above, a plurality of genes are related with oxygen resistance; that is, it is not the case that only a single gene is responsible for imparting oxygen resistance to a microorganism. Therefore, difficulty is encountered in practically using it.
It has been also reported by the inventors of the present invention that fnr gene present in Lactobacillus casei and Lactobacillus rhamnosus is a particularly required gene for bacterial growth under an aerobic condition, and it is also an oxygen resistance-imparting gene (Patent Document 2).
However, regarding an oxidative stress, resistance to hydrogen peroxide generated during metabolism is also important. In this regard, although there has been a report about a gene involved with elimination of hydrogen peroxide such as NADH peroxidase or catalase in microorganisms of genus Lactobacillus, a gene directly exhibiting hydrogen peroxide resistance has not yet been identified.